The mechanism by which insulin is able to activate pyruvate dehydrogenase in rat epididymal adipose tissue will be investigated. This activation process is of especial interest because pyruvate dehydrogenase is present within the confines of the mitochondrial membranes, yet insulin apparently need not enter fat cells in order to exert its effects. Therefore it is postulated that a cytoplasmic messenger mediates this effect of insulin. It is possible that many and perhaps all actions of insulin are mediated by cytoplasmic messengers and the investigation of this system may therefore offer a broad insight into the mechanism of action of this hormone. Pyruvate dehydrogenase activity is regulated by a phosphorylation-dephosphorylation cycle with only the dephospho-form active. Experiments will be carried out to determine whether insulin activates the phosphatase or inactivates the kinase involved. Intact fat cell mitochondria will be employed in a broad-ranging investigation of the role of cytoplasmic factors in the regulation of pyruvate dehydrogenase. In particular the possible presence of a unique cytoplasmic messenger of insulin's action will be sought. Further attention will be given to the role of cyclic-nucleotides in mediating this effect of insulin. Studies of the inactivation of the hormone-sensitive lipase will also be conducted. The goal will be to characterize the chemistry involved and to elucidate the properties of the inactivating enzyme. The ability of insulin to activate the cyclic-AMP phosphodiesterase will also be studied.